Kiran Kamran  ( Department of Anatomy, Fazaia Medical College, Air University, Islamabad. )
Muhammad Yunus Khan  ( Department of Anatomy, CPSP Regional Center, Islamabad, Pakistan. )
Liaqat Ali Minhas  ( Department of Anatomy, Nothern Border University, Arar, Saudi Arabia. )

Objective: To study the effects of ethanol vapour exposure on atrial and ventricular walls of heart in chick embryo.
Methods: The study design was experimental, conducted at Islamabad Centre of College of Physicians and Surgeons, Pakistan. One hundred and eighty chicken eggs were divided into two groups, experimental and control, of 90 eggs each. Each group was subdivided into three subgroups of 30 eggs each based on the day of sacrifice. Experimental group was exposed to ethanol vapours and then compared with age matched controls.
Results: The thickness of atrial and ventricular walls along with lengths of valvular cusps increased in hearts of day 7 and day 10 chick embryos in experimental group. There was thinning of walls and decreased length of valvular cusps in hearts of experimental chicks on hatching as compared to age matched controls.
Conclusion: Ethanol vapour exposure during development causes cardiac and septal wall thickening during initial days of development followed by cardiac and septal wall thinning which is a classical picture of alcohol induced cardiomyopathies.
Keywords: Ethanol vapour, chick embryo, atrial and ventricular walls. (JPMA 66: 1227; 2016)

Introduction
Alcohol drinking has ill-effects on health of humans. Alcohol misuse results in damage to almost every organ system of the body potentially resulting in serious illnesses like liver disease¹ and alcoholic brain disease.² Prenatal alcohol exposure can lead to foetal abnormalities. Alcohol intake exerts teratogenic effects on the developing foetus known as foetal alcohol syndrome.³ Alcohol drinking also impedes the functions of the heart, a condition referred to as alcoholic cardiomyopathy.⁴
Currently another method of alcohol abuse has been utilized through a machine known as" Alcohol without Liquid Vaporizer" which allows users to inhale alcohol vapours instead of drinking alcohol.⁵ Another device is used in Canada for the same purpose named as "Vaportini". The two apparatuses have different methodologies but serve the same purpose that is inhaling alcohol vapours rather than drinking. Product manufacturers emphasize that vapour inhalation does not have any ill effects on health like drinking alcohol but researchers are of the opinion that inhaling alcohol vapours bypasses its passage through stomach and delivers alcohol directly to the brain.⁶ Various studies are now in progress, in which animal models are exposed to ethanol vapours resulting in serious consequences.⁷ The effects of ethanol vapour exposure during gestation have also been under research lately to see whether ethanol vapour exposure has same effects on the foetal heart as drinking ethanol.⁸ This research was designed to study the effects of ethanol vapour exposure on atrial and ventricular walls of chick embryo.

Materials and Methods
This research was conducted at Department of Anatomy, College of Physicians and Surgeons, Islamabad from 2006 to 2007. The design of this study was experimental. One hundred and eighty Desi Chicken eggs were divided into an experimental group B and control group A comprising 90 eggs each. Each group was further subdivided into three subgroups 1, 2 and 3 containing 30 eggs each. Subgroup 1 was sacrificed on day 7, subgroup 2 on day 10 of incubation and subgroup 3 on hatching or day 22 whichever was earlier.  The day when eggs were placed in the incubator was taken as day 1. The temperature was maintained at 102°F and the relative humidity was kept between 70% and 80%.⁹

Group B was exposed to ethanol vapours produced in a tightly sealed glass chamber. The level of ethanol vapours was monitored and maintained with the help of a breathalyzer.⁷ Range of ethanol vapours was kept between 0.75mg/l to1.5mg/l. Subgroup B1 was exposed to ethanol vapours from day 1 to day 6. Subgroup B2 and B3 were exposed to ethanol vapours from day 1 to day 9.
Chick embryos were sacrificed on their respective days and hearts were processed for paraffin embedding. Serial sections of 15 to 20um thickness were taken and stained with haematoxylin and eosin to measure the atrial, ventricular and septal wall thickness. The lengths of right and left atrioventricular valvular cusps were also measured.

The thickness of interatrial and interventricular septa was measured by taking the maximum transverse distance between two points on the septal walls. The thickness of left and right atrial and ventricular walls was measured by taking the maximum transverse distance between two points on the respective walls. Right atrioventricular valve has only one cusp. Length of right atrioventricular valve cusp was measured by taking the distance between the free and attached end of the cusp. Left atrioventricular valve has two cusps. Length of left atrioventricular valve cusps was measured by taking the distance between attached and free end of the respective cusp. (Figure-1, 2)

The data was analyzed statistically using SPSS version 10. Student t test was applied to the quantitative data.

Results
The thickness of interatrial and interventricular septa of experimental group B1 was more than that of control group A1.This difference was not statistically significant for interatrial septa but was significant in case of interventricular septa (Table-1).
The thickness of right ventricular and left atrial wall of experimental group B1 was more than that of control group A1. This difference was not statistically significant for left atrial wall but was significant in case of right ventricular wall. The thickness of left ventricular wall and right atrial wall of control group A1was more than that of experimental group B1 but this difference was statistically not significant (Table-1). The length of right atrioventricular valve was more in control group A1 than that of experimental group B1; however this difference was not statistically significant. The length of right cusp of left atrioventricular valve was significantly greater in experimental group B1 than that of control group A1. The length of left cusp of left atrioventricular valve was more in control group A1 than in experimental group B1; however this difference was not statistically significant (Table-1)

The thickness of interatrial and interventricular septa of experimental group B2 was more than that of control group A2; however the difference was statistically not significant (Table-2). The thickness of left atrial wall and right ventricular wall was significantly more in experimental group B2 than that of control group A2. The thickness of right atrial wall was more in experimental group B2 than that of control group A2 but the difference was not statistically significant. The thickness of left ventricular wall was more in control group A2 than that of experimental group B2 but this difference was not statistically significant (Table-2). The length of right atrioventricular valve and left cusp of left atrioventricular valve was significantly more in experimental group B2 than that of control group A2. The length of right cusp of left atrioventricular valve was more in experimental group B2 than that of control group A2 but this difference was not statistically significant (Table-2)

The thickness of interatrial septum of newly hatched chicks of control group A3 was significantly more than that of experimental group B3. The thickness of interventricular septum of newly hatched chicks of control group A3 was more than that of experimental group B3; however, this difference was not statistically significant. The thickness of left and right atrial wall was more in control group A3 than experimental group B3 but these differences were not statistically significant (Table-3)

The thickness of left ventricular wall and right ventricular wall was also more in control group A3 than that of experimental group B3 but again these differences were not statistically significant (Table-3). The length of right atrioventricular valve and left cusp of left atrioventricular valve was more in control group A3 than that of experimental group B3; however, this difference was not statistically significant. The length of right cusp of left atrioventricular valve was significantly more in control group A3 than that of experimental group B3 (Table-3). No identifiable anomaly was seen.

Discussion
Interventricular, interatrial and septal thickness increased in alcoholic chicks at day 7 showing enhanced growth. The length of right atrioventricular (AV) valve and left cusp of left atrioventricular valve (AV) were longer in chick embryos belonging to control group as compared to those in experimental group but this was not statistically significant.

The hearts of ethanol-exposed chick embryos at day 10 had increased thickness of walls and septa. The valves were also longer in length in experimental group as compared to the control group which depicted enhanced growth.

The thickness of interatrial septum of newly hatched chicks of control group A3 was significantly more than that of experimental group B3. The thickness of interventricular septum of newly hatched chicks of control group A3 was more than that of experimental group B3; however, this difference was not statistically significant. In the present study, the length of right atrioventricular valve and both the cusps of left atrioventricular valve was less in the experimental group of B3 as compared to control group A3 again showing depressed growth.

 
The thickness of atrioventricular walls and septa increased in experimental groups during initial ten days of incubation. The thickness of atrioventricular walls and septa was less in experimental group B3 as compared to the control A3 group showing that the experimental hearts had thinning of their walls. This is in accordance with the previous studies which show that initial response to drinking was an increased thickness of the chamber walls followed later on by thinning of walls creating a classical picture of alcoholic cardiomyopathy.¹⁰

It is seen in previous researches that Alcoholic Heart Muscle Disease (AHMD) occurs in phases, starting with an asymptomatic phase, succeeding to a symptomatic phase and ultimately heart failure. In general, initial signs of Alcoholic cardiomyopathy (ACM) appear to be increased left ventricular mass along with increased posterior and septal wall thickening. This was in accordance with the findings of this study in which on day 7 and 10 the alcoholic heart had increased in thickness of atrial, ventricular and septal walls but this was statistically not significant. After day 10 alcohol was withdrawn, and on hatching it was seen that the alcoholic hearts had wall thinning as compared to controls which is the classical picture of alcoholic cardiomyopathy. These findings were consistent with earlier work on alcoholic cardiomyopathies.¹¹ Alcohol causes widening of heart chambers, cardiac muscle cell hypertrophy, reduced ventricular contractility, disorganized myofibrillary architecture and diminished ejection volumes.¹²

So even after ethanol withdrawal the alcoholic heart is in a compromised state with decreased myocardial contractility due to disorganized myocardial architecture. Reduced ejection fraction enhances end systolic volume, ¹³ increasing volume overload resulting in dilatation of heart chambers as well as cardiac wall thinning.

Another possible cause of cardiac wall thinning on hatching could be ethanol withdrawal after day 10. Withdrawal has damaging effects which might have resulted in depression of growth of the experimental hearts leading to cardiac wall thinning. It was seen in certain studies that chronic exposure to ethanol and sudden ethanol withdrawal produced toxic effects and oxidative stress leading to neuronal death in rat brains.¹⁴ Substantial losses of hippocampal neurons have also been noted in rat brains at various times following ethanol withdrawal.¹⁵ Chronic exposure to ethanol and its withdrawal produces an increase in reactive oxygen species triggering activation of protein kinase C. This later on aggravates apoptotic events producing DNA breakup¹⁴ ultimately causing tissue and organ damage. It has been shown that ethanol-induced apoptosis is probably the basic mechanism underlying ethanol-induced disorders such as foetal alcohol syndrome. Both humans and animal models of alcohol cardiomyopathy show that myocyte loss is the major etiological factor underlying alcohol induced cardiac dysfunction.¹⁶

There are many studies verifying the adverse effects of chronic alcohol consumption on the function of mitochondria and sarcoplasmic reticulum function which could be another cause of alcoholic cardiomyopathy.^17,18 Alcohol decreases the amount of protein synthesis in cardiac muscle which lessens the rate of contraction¹⁹ thus enhancing end systolic volume ultimately leading to cardiac wall thinning. Alcohol also decreases the rate of contraction by interfering with excitation contraction coupling at the muscle cell membrane by disturbing the calcium transients.²⁰

Persistent raised levels of norepinephrine produce adverse effects on the myocardium, some of which include myocyte hypertrophy, toxicity, and apoptosis. Adams and Hirst demonstrated in male rats that alcohol intoxication for 2 to 4 days (via gastric intubation) was accompanied by marked increase in urinary norepinephrine and epinephrine. These researchers proposed a direct role of catecholamines in alcohol-induced myocytehypertrophy.²¹ With marked ethanol consumption, there is higher occurrence for hypertension, cardiomyopathy, and arrhythmias.²²
This study shows that ethanol vapour exposure is as hazardous to developing heart as drinking alcohol.

Conclusion
Ethanol vapour exposure during development causes cardiac and septal wall thickening during initial days of development followed by cardiac and septal wall thinning which is a classical picture of alcohol induced cardiomyopathies.

Disclosure: No.

Conflict of Interest: No.

Funding Sources: No.

References
1.DU ZY, Gao J, Wang WX, Yu CH, Dou XB.[Synergistic action of 4-hydroxynonenal and tumor necrosis factor involving the NF-kB/IkBa signaling pathway in alcohol-induced liver injury].ZhonghuaGanZang Bing ZaZhi. 2013 ;21:747-52. doi: 10.3760/cma.j.issn.1007-3418.2013.10.007. Chinese.
2.Kroenke CD, Rohlfing T, Park B, Sullivan EV, Pfefferbaum A, Grant KA. Monkeys that Voluntarily and Chronically Drink Alcohol Damage their Brains: a Longitudinal MRI Study. Neuropsychopharmacology. 2014; 39: 823-30. doi: 10.1038/npp.2013.259.
3.Landgraf MN, Nothacker M, Kopp IB, Heinen F. The Diagnosis of Fetal Alcohol Syndrome.DtschArztebl Int. 2013;110:703-710. Epub 2013 Oct 18. Review.
4.George A, Figueredo VM. Alcoholic cardiomyopathy: a review. J Card Fail. 2011 ;17:844-9. doi: 10.1016/j.cardfail.2011.05.008. Epub 2011 Jun 16.
5.Wikipedia The Free Encyclopedia: Alcohol Inhalation [online] updated 30/6/2014 [cited 2014 July 22]. Available from URL: http://en.wikipedia.org/wiki/Alcohol_without_liquid
6.Maclean\\\'s: Vaportini doesn\\\'t live up to the hype [online] updated 6/7/2013 [cited 2015 June 30] Available from URL: http://www.macleans.ca/society/life/take-a-deep-breath-and-dont-drive/
7.Rouach H, Andraud E, Aufrère G, Beaugé F. The effects of acetaldehyde in vitro on proteasome activities and its potential involvement after alcoholization of rats by inhalation of ethanol vapours. Alcohol Alcohol. 2005;40:359-66. Epub 2005 Jun 27.
8.Kamran K, Khan MY, Minhas LA. Ethanol vapour induced dilated cardiomyopathy in chick embryos. J Pak Med Assoc. 2013; 63:1084-8.
9.Kamran K, Khan MY, Minhas LA. Effects of ethanol vapour exposure on survival of chick embryos.JColl Physicians Surg Pak 2009; 19: 150-3. doi:03.2009/JCPSP.150153
10.Spies CD, Sander M, Stangl K, Fernandez-Sola J, Preedy VR, Rubin E et al. Effects of alcohol on the heart. Curr Opin Crit Care. 2001;7:337-43.
11.McConnell BK, Jones KA, Fatkin D, Arroyo LH, Lee RT, Aristizabal O, et al. Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice.J Clin Invest. 1999; 104: 1771.
12.Aberle II NS, Ren J. Experimental assessment of the role of acetaldehyde in alcoholic cardiomyopathy. Biol Proced Online. 2003; 5:1-12.
13.Morris N, Kim CS, Doye AA, Hajjar RJ, Laste N, Gwathmey JK. A pilot study of a new chicken model of alcohol-induced cardiomyopathy.Alcohol Clin Exp Res. 1999; 23:1668-72.
14.Jung ME, Gatch MB, Simpkins JW. Estrogen neuroprotection against the neurotoxic effects of ethanol withdrawal: potential mechanisms.Exp Biol Med (Maywood).  2005; 230: 8-22.
15.Bengoechea O, Spies CD, Sander M, Stangl K, Fernandez-Sola J, Preedy VR, Rubin E et al. Effects of alcohol on the heart. Curr Opin Crit Care. 2001 ;7:337-43.
16.Cartwright MM, Smith SM. Increased cell death and reduced neural crest cell numbers in ethanol-exposed embryos: partial basis for the fetal alcohol syndrome phenotype. Alcohol Clin Exp Res. 1995; 19: 378-86.
17.Alexander CS. Electron microscopic observations in alcoholic heart muscle disease. Br Heart J. 1967; 29: 200-6.
18.Tsiplenkova VG, Vikhert AM, Cherpachenko NM. Ultrastructural and histochemical observations in human and experimental alcoholic cardiomyopathy. J Am Coll Cardiol. 1986; 8:27A-32A.
19.Vary TC, Deiter G. Long-term alcohol administration inhibits synthesis of both myofibrillar and sarcoplasmic proteins in heart.Metabolism. 2005; 54: 212-9.
20.Ponnappa BC, Rubin E. Modeling alcohol\\\'s effects on organs in animal models. Alcohol Res Health. 2000; 24: 93-104.
21.Adams MA, Hirst M. Adrenal and urinary catecholamines during and after severe ethanol intoxication in rats: a profile of changes. Pharmacol Biochem Behav. 1984; 21: 125-31.
22.Kodavali L, Townsend RR. Alcohol and its relationship to blood pressure. Curr Hypertens Rep. 2006; 8: 338-44.